Method for detecting viable cells in a sample by using a virus

ABSTRACT

A method for detecting viable cells such as bacterial cells, within a sample, said method comprising (i) incubating said sample with a virus which is able to infect said cells under conditions which allow said virus to infect and replicate within any such cells which are viable; and (ii) detecting any nucleic acid obtained by replication of the virus in said cell.

This application is a divisional application of U.S. patent applicationSer. No. 11/816,331, now U.S. Pat. No. 8,071,337, filed Nov. 9, 2007,which is a 371 national stage application of PCT/GB06/000551, filed Feb.16, 2006, each of which is hereby incorporated by reference in itsentirety.

The entire contents of a paper copy of the “Sequence Listing” and acomputer readable form of the sequence listing, entitled091008_Seq_List.txt that is 1 kilobytes in size and created on Nov. 24,2009, are herein incorporated by reference.

The present application relates to a method for detecting the presenceof viable cells within a sample, as well as to kits for use in themethod.

The rapid detection of bacteria and other organisms is very important inthe field of public safety. It is particularly important in the foodindustry where it is essential that food and drinks are free fromcontamination by harmful bacteria and other microorganisms, for exampleE. coli 0157.

It is also very important in the field of veterinary and medicaldiagnostics that potentially harmful bacteria and other organisms areidentified quickly, to allow infection to be controlled and to enablethe correct treatment to be given to the animal or patient. It is alsoimportant that medical, cosmetic and veterinary preparations are freefrom harmful contaminants such as bacteria.

In addition some bacteria and other organisms, for example, Bacillusanthracis have potential for use as biological warfare agents. It istherefore important that the presence of such organisms in a sample canbe tested for quickly. For example, it has been estimated that there isonly a 6-hour window in which to administer effective treatment afterthe initial inhalation of Bacillus anthracis spores.

Several methods are currently available for detection of bacteria andother organisms.

For example traditional culture-based methods can be used to detect thepresence of bacteria in a sample. However a problem commonly encounteredin such assays is that the concentration of the organism in a sample isgenerally very low. Thus it may be necessary to incubate the sample fora significant period of time prior to conducting the assay in order toculture the organism to detectable levels. This delay may beunacceptable in many situations where public health is at risk.

Alternatively PCR and other nucleic amplification technologies provide afast and sensitive DNA based means of detecting the presence of bacteriaand other organisms. These techniques in general rely on the detectionof bacterial DNA using amplification reactions such as the polymerasechain reaction (PCR).

However there are disadvantages associated with such amplificationtechniques because DNA is a very robust chemical species and can surviveintact when the host cell is dead.

For example, if a poultry product has been contaminated with apathogenic bacterium such as Salmonella and then cooked, the food willhave been rendered safe. In a traditional culture-based method ofdetection the food will be shown to be safe but the method itself willtake a long time to conduct, whereas a DNA method whilst quicker willshow a false positive result because the Salmonella DNA will still bepresent in the sample.

Alternatively WO 2003/035889 describes a technique in which modifiedphage comprising marker sequences are used to detect viable cells. Inthis method the modified phage are introduced into a sample to betested. If viable cells are present, then the phage can replicate usingthe cells machinery resulting in a detectable increase in the markersequence.

However there are disadvantages associated with this technique.

Firstly the need to incorporate marker sequences necessarily increasesthe complexity of the test, since the phage have to be specificallyengineered to contain the desired marker sequence. Such modified phagewill therefore increase the cost and complexity of such tests.

In addition since the marker sequence is added to the sample to betested, due to its presence in the phage, it is necessary toquantitatively detect any increase in the amount of this marker sequencein the sample. This means that the incubation period has to be longenough to ensure that false positive results are not generated.

There is therefore a need to provide a reliable test method forpotential biological hazards, which can be operated easily, and reliablywith a low level of false positive or negative results.

Accordingly the present invention provides a method for detecting viablecells within a sample, said method comprising (i) incubating said samplewith a virus which is able to infect said cells under conditions whichallow said virus to infect and replicate within any such cells which areviable; and (ii) detecting any nucleic acid obtained by replication ofthe virus in said cell.

By detecting nucleic acid obtained specifically by replication of thevirus, it is possible to determine that there are viable cells presentin the sample, as compared simply to dead cells.

In a particular embodiment the invention provides a method for detectingviable cells within a sample, said method comprising (i) incubating saidsample with a virus which is able to infect said cells under conditionswhich allow said virus to infect and replicate within any such cellswhich are viable; and (ii) detecting any nucleic acid obtained byreplication of the virus in said cell, provided that when the virus is amodified virus which has been modified to include a marker sequence, thenucleic acid detected is one which is uniquely produced on replicationof the virus within the cell.

In order to ensure that any viable cells are infected during step (i),they may be subjected to a pre-conditioning step, for example bybringing them to an incubation temperature of for example 37° C.-42° C.,for a suitable period of time, for example for 2.5-4.5 hours beforeaddition of the virus. This may be particularly necessary if forinstance, the sample contains stressed bacteria, for example as a resultof storage or isolation procedures such as immunomagnetic separation(IMS) methods. They may then be held at the incubation temperature for aperiod of time to allow infection and some replication to occur.

The virus used may be a single stranded (ss) or double stranded (ds) RNAor DNA virus.

The nucleic acid detected in step (ii) may occur within the virusitself. In this case, it is necessary to quantitatively detect anincrease in the amount of this nucleic acid in the sample. This methodis advantageous over that described in WO 03/035889 since there is noneed to modify the virus to include a marker sequence.

Preferably, however the nucleic acid detected in step (ii) is notcontained within the virus itself, and is uniquely produced onreplication of the virus within the cell. This means that the detectionof any nucleic acid having this particular sequence would be indicativeof the presence of viable cells within the sample, as they have beenutilized in the production of the virus.

The virus may be either a wild type virus or a modified virus. When thevirus is a modified virus which has been modified to include a markersequence, the nucleic acid detected is one which is uniquely produced onreplication of the virus within the cell.

In a particular embodiment the virus is not modified to include anymarker sequence(s), for example target repeat sequences.

The virus may however be modified for other purposes, for example toincrease the infectivity or specificity of the virus. Such viruses maybe commercially available and therefore can provide advantages overnaturally occurring viruses without significantly increasing thecomplexity or cost of the method, or they may be produced usingconventional transformation methods.

The detection option which is selected will depend upon the particularvirus being used.

In a particular embodiment, the virus is an RNA virus, in particular asingle stranded RNA virus.

Particularly preferred single stranded RNA virus are Class VI RNAviruses, which comprise a single positive RNA strand. These virusesconvert their RNA into complementary DNA (cDNA) using a process calledreverse transcription. Converting RNA into cDNA enables the virus toreplicate inside the host cell.

However, detection of the cDNA produced in step (ii) above, provides aunique indicator that viable cells, capable of being infected andsustaining a viral infection, are present within the sample.

The reverse transcriptase enzyme necessary for carrying out reversetranscription is an enzyme which is often present in the virion andtherefore the virus does not need to rely on the host cell to providethis essential enzyme. However the host must provide many other factorsthat the virus needs to be able to infect and replicate within the hostcell.

For example internalization of the virus into the host cell requireshost factors such as surface proteins and cell membranes with which thevirus must interact. In addition reverse transcription requires a stablemilieu containing the necessary dNTP substrates to form cDNA from theviral RNA.

If the host cell is not viable and is therefore not producing theadditional factors such as those described above, then the virus willnot be able to infect and replicate within the host cell, and therebygenerate detectable DNA which may for instance, form the target of anamplification reaction.

If the host cell is viable, the RNA virus will be able to infect andreplicate within the host cell producing detectable cDNA. If the hostcell is not viable then the virus will not be able to infect the hostcell and/or replicate inside it and so no cDNA will be detectable.

In this embodiment of the invention, during step (i) it is necessaryonly to incubate the sample for a period long enough to ensure that atleast one cDNA is produced by the host cell. That can then be detectedusing a conventional amplification reaction such as PCR. Howeverincubation for a period long enough to produce multiple copies may alsobe carried out. Generally, incubation periods of from 30 minutes to 1hour would be sufficient to generate detectable nucleic acid.

Alternatively, the virus may comprise a single negative strand RNAmolecule, such as class V viruses. On infecting a viable host cell, thecell will produce a complementary positive RNA strand, specifically anmRNA strand. As described above however, this process can only takeplace within the stable milieu of an infected cell. The thus-producedpositive RNA strand may then be detected using for example aconventional reverse transcriptase-PCR reaction (RT-PCR).

Preferably, the detection is carried out using a single primer in the RTstep so that only a single complementary DNA molecule is produced.

In a particular embodiment, this cDNA is then detected by a conventionalPCR amplification, designed to amplify a region within the cDNA.Digestion of the RNA strand prior to this amplification may bepreferable in order to ensure that contamination is minimized.

Alternatively, an increase in the amount of viral nucleic acid itselfcan be used as an indicator of the presence of viable cells. Again, thisis because replication of the virus can take place only within a viablecell. In this embodiment the nucleic acid to be detected is other than areporter sequence, and is preferably a wild-type sequence.

For instance, where the virus is a DNA virus, such as a single strandedclass II virus, or a double-stranded class I virus, it will generally benecessary to detect an overall increase in DNA following infection todetermine that specifically viable cells are present within the sample.This can be done in various ways, but probably the most convenient wouldbe to amplify the DNA using a quantitive PCR assay, such as the TAQMAN™assay or the like.

Where the virus is an RNA virus, an increase in RNA may need to bedetected, for instance using RT-PCR.

The sample to be tested in the method of the invention may be anysample, which is suspected of or known to contain prokaryotic cellsand/or lower eukaryotic cells and/or higher eukaryotic cells.

Examples of samples included within the scope of this invention include,food samples, samples of human or animal bodily fluids or tissues and inparticular clinical samples, medicinal, cosmetic or veterinarypreparations or medications, plant samples, soil samples, air samples,water samples and cell culture samples. Most preferably the sample is afood sample.

This method of the invention can be applied to detect viable examples ofany cell that a virus is capable of infecting. These include prokaryoticand eukaryotic cells. The cells may be for example microorganisms, suchas bacteria. Particular examples of bacteria which may be detectedinclude E. coli, Salmonella, Listeria, Campylobacter, Legionella,Mycobacterium, Staphylococcus or Streptococcus.

Alternatively the cells may be contained within small organisms, such asinsects, plants or fungi.

Additionally they may be present in cells or cell lines includingmammalian cell lines, plant and fungal cell lines. Detection of viablecells in cell lines is frequently carried out, for instance during thescreening of pharmaceutical or other reagents, or in biologicalanalysis, and the method described herein may be useful in this context.

The method may be particularly useful for example in analyzing cellcontaining samples from patients who have been treated for a particularbacterial infection with antibiotics. Appropriate samples taken fromthese patients can be tested in accordance with the method describedherein to determine whether the antibiotic treatment has been effectiveor not.

The virus can be either a wild type virus or a recombinant virus. If thecell is viable, the virus will be able to infect it and replicate,either in the case of positive RNA, by producing a cDNA, or in othercases by producing more RNA, and in particular mRNA. Where a modifiedvirus is used detection of the complementary sequence of for example anintroduced marker sequence uniquely produced on replication of the ofthe virus within the cell, may then provide a clear indication of thepresence of viable cells. Suitable marker sequences may includeantibiotic resistance genes.

The method described above is general in nature in that it can be usedto detect the viability of any type of cell, which may be infected by avirus. For example if a sample needs to be tested for viable E. coli0157 cells any virus capable of infecting E. coli 0157 cells could beused.

In some samples for example a milk or soil sample, the number of typesof bacteria or other cells present may be very high. Testing for theviability of a particular type of cell in such samples is understandablydifficult. However in a preferred embodiment of this invention the virusused is specific to the type of cell, the presence of which, in viableform, is under investigation. In other words in this embodiment thevirus used must only be capable of infecting and replicating within thecell type under investigation.

For instance, a known coliphage is obtainable from the NationalCollection of Industrial and Marine Bacteria, Aberdeen, UK, as NCIMB10359. The Felix 01 phage obtainable from Diagnostics Pasteur (Sanofi)Watford UK is capable of infecting Salmonella strains, in particularSalmonella Newport.

In this preferred embodiment the specific virus used, will only producedetectable nucleic acid if the cell being tested for is present andviable no matter how may other types of bacteria or other cells arepresent. This advantageously means that the cells to be tested do nothave to be isolated before their presence in viable form can bedetermined.

Viruses may be used singly or as multi-specificity mixtures, where theviability of more than one cell is being looked for. In the latter case,detection of multiple nucleic acid sequences, such as cDNA sequences,each characteristic of individual viruses is carried out subsequently.

The infection and replication cycle of viruses will often result in aninfected cell bursting and releasing its contents. If this is the case,the nucleic acid detected in step (ii) of the method described above canbe detected without actively lysing or homogenizing the cells. Howeverlysis or homogenization of any cells within the sample can be carriedout before step (ii) if desired or necessary.

Lysis or homogenization can be affected using any conventional method,for instance, by heating or example to 94° C., sonication or by additionof a lytic agent such as a detergent.

In a preferred embodiment, where for example a cDNA produced by thevirus or a DNA derived from a replicated RNA is detected in step (ii),this is preferably achieved using an amplification reaction, for examplea polymerase chain reaction (PCR). In this case, reagents, includingprimers, polymerases, nucleotides, and buffers as are well known, areadded at the end of step (i) after lysis of the cells if necessary, andthen subjected to thermal cycling as is conventional, in order toamplify any target DNA present.

The amplification product may then be detected using conventionalmethods such as gel electrophoresis, followed by visualization usingdyes.

Preferably the amplification reaction is carried out in such a way thatthe amplification product generates a detectable signal, and inparticular a visible signal, for example a fluorescent signal, as itprogresses. Many assay formats that produce such signals are known inthe art. They may utilize reagents such as DNA binding agents such asintercalating dyes which emit radiation and particularly fluorescentradiation at greater intensity when they are intercalated into DNA, aswell as probes and primers which include fluorescent labels, arranged toundergo fluorescent energy transfer (FET) and particularly fluorescentresonant energy transfer (FRET).

There are two commonly used types of FET or FRET probes, those usinghydrolysis of nucleic acid probes to separate donor from acceptor, andthose using hybridization to alter the spatial relationship of donor andacceptor molecules.

Hydrolysis probes are commercially available as TaqMan™ probes. Theseconsist of DNA oligonucleotides that are labelled with donor andacceptor molecules. The probes are designed to bind to a specific regionon one strand of a PCR product. Following annealing of the PCR primer tothis strand, Taq enzyme extends the DNA with 5′ to 3′ polymeraseactivity. Taq enzyme also exhibits 5′ to 3′ exonuclease activity.TaqMan™ probes are protected at the 3′ end by phosphorylation to preventthem from priming Taq extension. If the TaqMan™ probe is hybridized tothe product strand, an extending Taq molecule may also hydrolyze theprobe, liberating the donor from acceptor as the basis of detection. Thesignal in this instance is cumulative, the concentration of free donorand acceptor molecules increasing with each cycle of the amplificationreaction.

Hybridization probes are available in a number of forms. Molecularbeacons are oligonucleotides that have complementary 5′ and 3′ sequencessuch that they form hairpin loops. Terminal fluorescent labels are inclose proximity for FRET to occur when the hairpin structure is formed.Following hybridization of molecular beacons to a complementary sequencethe fluorescent labels are separated, so FRET does not occur, and thisforms the basis of detection.

Pairs of labelled oligonucleotides may also be used. These hybridize inclose proximity on a PCR product strand-bringing donor and acceptormolecules together so that FRET can occur. Enhanced FRET is the basis ofdetection. Variants of this type include using a labelled amplificationprimer with a single adjacent probe.

Other methods for detecting amplification reactions as they occur areknown however, and any of these may be used. Particular examples of suchmethods are described for example in WO 99/28500, British Patent No.2,338,301, WO 99/28501 and WO 99/42611.

WO 99/28500 describes a very successful assay for detecting the presenceof a target nucleic acid sequence in a sample. In this method, a DNAduplex binding agent and a probe specific for said target sequence isadded to the sample. The probe comprises a reactive molecule able toabsorb fluorescence from or donate fluorescent energy to said DNA duplexbinding agent. This mixture is then subjected to an amplificationreaction in which target nucleic acid is amplified, and conditions areinduced either during or after the amplification process in which theprobe hybridizes to the target sequence. Fluorescence from said sampleis monitored.

An alternative form of this assay, which utilizes a DNA duplex bindingagent which can absorb fluorescent energy from the fluorescent label onthe probe but which does not emit visible light, is described inco-pending British Patent Application No. 223563.8. Any of these assaysmay be used in the context of the method of the invention in order todetect the target cDNA sequence.

Many of these assays can be carried out in a quantitative manner as iswell known in the art, for example by monitoring the signal from theamplification mixture at least once during each cycle of theamplification reaction.

By carrying out the reaction in this way, the amount of nucleic acidpresent in the sample may be determined. This may be related to theamount of viable cells in the original sample, or it may be used todetermine an increase in the amount of the nucleic acid as a result ofviral replication within viable cells.

As stated above the particular nucleic acid detected may be anycharacteristic sequence, which is produced or replicated as a result ofviral infection of viable cells. Where single specificity viruses areused in the assay, this may be any sequence derived from the virus, orany other sequence introduced into a recombinant virus during itspreparation. When the virus is a modified virus which has been modifiedto include a marker sequence, the nucleic acid is one which is uniquelyproduced on replication of the virus within the cell.

Where multi-specificity mixtures of viruses are used in the method, thenit is necessary to detect nucleic acid sequences, which arecharacteristic sequences transcribed from each of the viruses used inorder to determine whether specific types of cells in the sample areviable. In this case therefore, it may be desirable to detect sequenceswhich are uniquely produced on replication of the virus within the celland correspond to specifically introduced marker sequences in each virusused.

In this case, where the sequences detected are DNA sequences, multiplexPCR reactions using different signalling reagents or systems may beemployed in order to detect the various sequences which are produced.This may be achieved, for example by labelling probes or primers used inthe amplification reaction using different labels, for example, labelswhich fluoresce at different wavelengths. Examination of the signal fromeach label, for example at each of the different wavelength, is thencarried out, if necessary with appropriate signal resolution where thewavelengths overlap.

Where, in the method of the invention, the nucleic acid being detectedis present in the virus itself, it is only an increase in the amount ofthat nucleic acid which can be used to indicate the presence of viablecells. Therefore, in step (ii) the sample is tested to detect anyincrease in the concentration of that nucleic acid sequence. An increasein the amount of the target nucleic acid sequence is indicative of thepresence of a viable host cell within the sample. Whereas no increase inthe amount of the target nucleic acid is indicative of the fact thatthere are no viable host cells in the sample being tested.

In this embodiment, it may be desirable to extract a sample from themixture formed by addition of virus to sample, prior to incubation, andthe amount of nucleic acid within this extracted sample tested, toprovide a baseline for comparison. Additional extracted samples may bealternatively or additionally taken at any time during incubation.

Most preferably the amount of nucleic acid present before and afterincubation is determined by a suitable method. For instance, DNAconcentrations may be detected using quantitative amplificationreactions, such as quantitive PCR, although other methods for detectingDNA which do not involve amplification may also be used. RNAconcentrations may be detected using RT-PCR, combined if appropriatewith PCR reactions to detect the thus produced DNA.

The pre-incubation sample and post-incubation sample must be treated inthe same manner so that the results can be directly compared. Mostpreferably a water control is also used.

A further aspect of the invention provides a kit for carrying out themethods described above. The kit suitably contains a virus, and one ormore reagents necessary for detecting a nucleic acid.

In a preferred embodiment the kit contains a virus, and one or morereagents necessary for detecting nucleic acid obtained by replication ofthe virus in said cell, provided that when the virus is a modified viruswhich has been modified to include a marker sequence, the one or morereagents are necessary for detecting a nucleic acid which is uniquelyproduced on replication of the virus within the cell.

Most preferably the virus is a bacteriophage however other DNA or RNAvirus, which is capable of infecting lower and/or higher eukaryoticcells may be used depending upon the nature of the cells under test.

Where the virus used is an RNA virus, this is preferably at leastpartially but preferably fully purified of contaminating DNA before step(i). Such viruses form a further aspect of the invention.

Suitably the kit may comprise an RNA virus and one or more reagents fordetecting cDNA obtainable by reverse transcription of a sequence withinsaid RNA virus. Most preferably the said one or more reagents comprisesa pair of primers which are specific for any cDNA obtainable by reversetranscription of a sequence within said RNA virus.

Alternatively the kit may comprise a DNA virus and one or more reagentssuitable for quantitatively detecting DNA. Most preferably the said oneor more reagents comprises a pair of primers which are specific for aDNA sequence in the virus.

Alternatively kits may comprise reagents such as primers, which arerequired to conduct an RT-PCR reaction to detect an RNA.

Possible additional elements of the kits comprise other reagentssuitable for use in the detection of the nucleic acid sequences. Inparticular, the kit may comprise intercalating dyes, or probes for usethe detection of any nucleic acids produced using the methods describedabove.

The primers may suitably be labelled in such a way that theamplification product is directly detectable. For example, they mayinclude fluorescent or other labels as described above.

Additionally or alternatively, the kits may include probes, which arespecific for the amplification product and which are labelled to assistin detection of product. They may comprise single- or dual-labelledhydrolysis or hybridization probes. When appropriate they may includeintercalating dyes or other DNA duplex binding agents, which formelements of the detection system.

The invention will now be particularly described by way of example withreference to the accompanying diagrammatic drawings in which:

FIG. 1 illustrates schematically, a detection method according to theinvention;

FIG. 2 illustrates amplification curves for all test samples in Example2 hereinafter;

FIG. 3 illustrates the results of an attempted amplification of any cDNAusing a forward primer showing that none of the test samples producedamplified cDNA;

FIG. 4 illustrates the results of an amplification of any cDNA using areverse primer showing that amplification was detected at approximatelycycle 25, 29, 32 and 34 for the neat, 1:10, 1:100 and 1:1000 test samplerespectively; and

FIG. 5 illustrates the results of an amplification of cDNA using bothforward and reverse primers, showing that amplification was detected atapproximately cycle 20, 23, 27 for the neat, 1:10 and 1:100 test samplerespectively.

EXAMPLE 1

In the embodiment illustrated in FIG. 1, a class V virus preparation,having a single stranded negative genome is applied to a sample.

The sample may be any suitable sample but may comprise a bacterialextract in broth or phosphate-buffered saline (PBS), wherein thebacteria are not stressed and therefore receptive to infection. Sampleswill be diluted if necessary, for example so that they may contain from6×10⁶ to 2×10³ cells ml⁻¹.

Virus preparations are suitably previously treated to removecontaminating DNA, for instance, by treatment with a DNAse enzyme. Theymay also be purified by centrifugation, with resuspension in for examplebuffered peptone water (BPW), for form phage titres of for instance 10¹⁰ml⁻¹.

These preparations, for instance about 20 μl phage, are then added,followed by gentle mixing for 30 seconds. Thereafter the mixture issuitably allowed to stand for 5 minutes to encourage phage attachment.

Incubation is then suitably carried out, for example in a water bath at37° C. for 30 minutes, during which the cultures may be gently stirred.

During this period, phage will infect viable cells (as illustrated) andmultiple copies of positive RNA are generated within those cells.

At the end of this procedure, if necessary the cells are lysed, forexample by heating them to 94° C., and the resultant mixture subjectedto an RT-PCR to generate the complementary DNA sequence from thepositive RNA. This is suitably done using a single primer, which isspecific for the positive RNA.

Example of typical conditions in a murine reverse transcriptase or AMVreaction are as follows:

-   -   Buffer: 10 mM Tris.HCl pH 8.3 (room temp), 1-6 mM MgCl₂, 0.1%        Gelatin (optional), 10-50 mM KCl, BSA Fraction V 1-100 μg/ml;    -   Nucleotides: 50-400 μM;    -   Primers: random hexanucleotides, specific primers (as in PCR),        or polydT primers (string of “T's”), 0.1-1 μM;    -   Enzyme: 0.25-5 units/μl (depending upon enzyme); and    -   Optional placental RNAse inhibitor, 1-20 units.

Treatment of the product with an RNAse will digest the RNA templateleaving only the cDNA, which can be amplified and detected using aconventional PCR reaction.

All these steps are quick and therefore provide a rapid assay fordetecting viable cells.

EXAMPLE 2 Strand-Selective MS2 RT-PCR Experiment

This experiment was conducted to show that cDNA can be produced anddetected using strand directed RT-PCR. Specifically the experimentinvestigated strand-specific two-step RT-PCR in MS2 bacteriophage. ThecDNA was made using the forward primer and reverse primer separately andusing both combined. As only one type of RNA is present in the MS2, onlyone of the primers (e.g. Forward or Reverse) was expected to produce DNAthat would amplify in a PCR.

Methods.

The primer and probe sequences used in the experiment indicated inTable 1. Both primers were diluted with DEPC treated H₂O to aconcentration of 10 μM. The probes were also diluted on to aconcentration of 2 μM. The first probe was diluted with 0.1×TE and thesecond probe was diluted with DEPC treated H₂O.

TABLE 1 MS2 Primers and Probes Modi- fica- Name Sequence (5′ to 3′)Length tion Forward  TCG TCG ACA ATG GCG  21 None Primer GAA CTG(SEQ ID NO: 1) Reverse  CTT TAG GCA CCT CGA  24 None Primer CTT TGA TGG(SEQ ID NO: 2) First  AGC TCT AAC TCG CGT TCA CAG  40 3′ FAM ProbeGCT TAC AAA GTA ACC TGT T (SEQ ID NO: 3) Second GTT CGT CAG AGC TCT GCG    33 5'Cy5 & Probe CAG AAT CGC AAA TAT  3'Phos- (SEQ ID NO: 4) phate

For the RT-step, three reaction mixes were prepared using theconcentration of reagents in Table 2:

TABLE 2 Reagents used in the RT step. Stock Amount added (μl) Final LotConcen- F & R F R Concen- Reagents No. tration Primers Primer Primertration Reaction 1009 5X 16 16 16 1X buffer dUTPs 24/11   2 mM 20 20 20 0.5 mM Forward — 10 μM 10 10 0 1.25 μM* Primer Reverse — 10 μM 10 0 101.25 μM* Primer *if added

The RT reaction mixes were made up to 71 μl with DEPC treated H₂O. Next,8 μl of MS2 (at concentration 4×10⁹/ml) was added to each tube. Thesamples were mixed and divided into two tubes containing 35 μl each, anddenatured at 90° C. for 10 min. The tubes were then placed on ice and0.5 μl of the RT enzyme MMuLV (60 units/μl, lot #173) was added to eachtube. The samples were mixed gently and placed in a 48° C. water bathfor 30 min for the transcription of cDNA.

The following test samples were prepared in duplicate for PCR analysis:

-   -   DEPC-treated H₂O, negative control (ntc)    -   4×10-fold dilution series of the cDNA made using the Forward        primer    -   4×10-fold dilution series of the cDNA made using the Reverse        primer    -   3×10-fold dilution series of the cDNA made using both primers    -   MS2 Purified DNA Product 1:1000 dilution, positive control

PCR reaction mixes were prepared using a Corbett robot. Theconcentrations of reagents in each reaction are listed in Table 3.

TABLE 3 Concentration of reagents used in the PCR mix. Stock Amountadded Final Reagents Lot # Concentration (μl) per sample ConcentrationTris Buffer 0016 0.5M 2 50 mM pH 8.8 BSA B8667 20 mg/ml 0.25 0.25 mg/mlMgCl₂ 019 100 mM 0.6 3 mM dUTPs 24/11 2 mM 2 0.2 mM First Probe — 2 μM 20.2 μM Second Probe — 2 μM 2 0.2 μM Forward Primer — 10 μM 2 1 μMReverse Primer — 10 μM 2 1 μM Taq Antibody GC102TA/7 5 units/μl 0.160.04 units/μl Taq Polymerase 14/4 5 units/μl 0.16 0.04 units/μl

The PCR mix (18 μl) was added to a LightCycler capillary along with 2 μlof test sample. The capillaries were capped and spun briefly to 3000rpm.

The following program was carried out on a LightCycler 1.0 (Roche):Amplification (50 cycles) of Denature at 95° C. for 5 s; Annealing at55° C. for 20 s (Single fluorescent reading); Extension at 74° C. for 5s; Melt (1 cycle); Hold at 50° C. for 15 s, Temp. Ramp at 95° C. at 0.1°C./s (Continuous fluorescent reading).

Data were analyzed using the LightCycler Data Analysis program.

The results are shown in FIGS. 2 to 5. FIG. 2 shows the results for alltest samples. FIG. 3 shows the results for test samples containing theforward primer. FIG. 4 shows the results for test samples containing thereverse primer. FIG. 5 shows the results for test samples containingboth primers.

Summary of Results:

Using the Forward primer resulted in insufficient cDNA for detection inPCR. However, the Reverse primer produced sufficient cDNA for detectionin PCR. When both primers were used cDNA was amplified with greaterefficiency and detected earlier in PCR amplification.

Thus it has been demonstrated that a cDNA uniquely produced by reversetranscription of an RNA can be specifically detected using stranddirected RT-PCR.

The invention claimed is:
 1. A method for detecting viable bacterialcells within a sample, the method comprising: i) incubating the samplewith a wild type virus specific for the cells, under conditions whichallow the virus to infect and replicate within any such cells which areviable; ii) detecting an increase in the amount of a nucleic acidobtained by replication of the virus in the cells using quantitativePolymerase Chain Reaction; and iii) correlating the increase in thenucleic acid detected in step (ii) with the presence of the viablebacterial cells in the sample.
 2. The method of claim 1 wherein thevirus is a DNA or RNA virus and the nucleic acid detected in step (ii)occurs within the virus.
 3. The method of claim 1, wherein beforedetection in step (ii) any intact cells within the sample are lysed. 4.The method of claim 1, wherein the sample is a food or clinical sample.5. The method of claim 1, wherein the virus is a bacteriophage.
 6. Themethod of claim 1, wherein the cell is an E. coli, Salmonella, Listeria,Campylobacter, Legionella, Mycobacterium, Staphylococcus orStreptococcus.